Angiotensin type 2 (AT2) receptor agonists and uses thereof

ABSTRACT

The invention relates to novel pharmaceutically-useful peptides, in particular cyclic peptides that are agonists of the AngII type 2 receptor (AT2 receptor). The invention further relates to the use of such peptides as medicaments, to pharmaceutical compositions containing them, and to their production.

This patent application is a divisional of U.S. application Ser. No.15/009,846, filed Jan. 29, 2016, now issued as U.S. Pat. No. 9,707,268,which is a divisional of U.S. application Ser. No. 13/988,085, filedOct. 17, 2013, now issued as U.S. Pat. No. 9,290,540, which is the U.S.National Stage of PCT/NL2011/050793, filed Nov. 22, 2011, which claimsthe benefit of priority from EP 10192208.6, filed Nov. 23, 2010, thecontents of each of which is incorporated by reference in its entirety.

FIELD

The invention relates to novel pharmaceutically-useful peptides, inparticular cyclic peptides that are agonists of the angiotensin II type2 receptor (hereinafter the AT2 receptor). The invention further relatesto the use of such peptides as medicaments, to pharmaceuticalcompositions containing them, and to synthetic routes to theirproduction.

BACKGROUND

The endogenous hormone AngII is a linear octapeptide(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) (SEQ ID NO:1), and is the activecomponent of the renin-angiotensin system (RAS). It is produced by thesequential processing of the pro-hormone angiotensinogen by renin andangiotensin converting enzyme (ACE). The RAS plays an important role inthe regulation of blood pressure, body fluid and electrolytehomeostasis. Ang II exerts these physiological actions in many organsincluding the kidneys, the adrenal glands, the heart, blood vessels, thebrain, the gastrointestinal tract and the reproductive organs (deGasparo et al, Pharmacol. Rev. (2000) 52, 415-472). Two main classes ofAngII receptors have been identified, and designated as the type 1receptor (hereinafter the AT1 receptor) and the AT2 receptor. The AT1receptor is expressed in most organs, and is believed to be responsiblefor the majority of the biological effects of AngII. The AT2 receptor ismore prevalent than the AT1 receptor in fetal tissues, the adultovaries, the adrenal medulla and the pancreas. An equal distribution isreported in the brain and uterus (Ardaillou, J. Am. Soc. Nephrol., 10,S30-39 (1999)). Several studies in adult individuals appear todemonstrate that, in the modulation of the response following AngIIstimulation, activation of the AT2 receptor has opposing effects tothose mediated by the AT1 receptor.

The AT2 receptor has also been shown to be involved in apoptosis andinhibition of cell proliferation (see de Gasparo et al, supra). Further,it seems to play a role in blood pressure control. The functionalrelevance of AT2 receptors in cardiovascular disease is discussed inJones et al. (Pharmacology & Therapeutics 120 (2008) 292-316). Theexpression of AT2 receptors has also been shown to increase duringpathological circumstances, such as vascular injury, wound healing andheart failure (see de Gasparo et al, supra).

The expected pharmacological effects of agonism of the AT2 receptor aredescribed generally in de Gasparo et al, supra. AT2 receptor agonistshave been shown to be of potential utility in the treatment and/orprophylaxis of disorders of the alimentary tract, such as dyspepsia andirritable bowel syndrome, as well as multiple organ failure (see WO99/43339).

AngII antagonists (which bind to the AT1 and/or AT2 receptors) have beendisclosed in inter alia European patent applications EP 409 332, EP 512675; international patent applications WO 94/27597, WO 94/02142, WO95/23792 and WO 94/03435; and U.S. Pat. Nos. 5,091,390, 5,177,074,5,412,097, 5,250,521, 5,260,285, 5,376,666, 5,252,574, 5,312,820,5,330,987, 5,166,206, 5,932,575 and 5,240,928. US 2009/326026 disclosesthe use of tricyclic, imidazole-containing compounds as AT2 agonist. WO2004046128 relates to bicyclic compounds which are useful as selectiveagonists of the AT2 receptor.

Peptide and non-peptide AT2 receptor agonists, unrelated structurally tothose described herein, and potential uses thereof, have been disclosedin, for example, international patent applications WO 00/38676, WO00/56345, WO 00/09144, WO 99/58140, WO 99/52540, WO 99/46285, WO99/45945, WO 99/42122, WO 99/40107, WO 99/40106, WO 99/39743, WO99/26644, WO 98/33813, WO 00/02905 and WO 99/46285; U.S. Pat. No.5,834,432; and Japanese patent application JP 143695.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. KcAng-(1-7) and YcAng-(1-7) are highly potent vasodilators.KcAng-(1-7) and YcAng-(1-7) induce vasodilation ofphenylephridine-precontracted rings of aorta of Sprague Dawley rat. x:control; □: native angiotensin-(1-7); ▪: cAng-(1-7); ◯: KcAng-(1-7); ●:YcAng-(1-7).

FIGS. 2A through 2C. N-extended cAng-(1-7) analogs require the 4,7thioether ring for agonistic action via the AT2 receptor.

cAng-(1-7) is a 4,7 thioether bridged Ang-(1-7). XcAng-(1-7), which iscAng-(1-7) with one variable additional amino acid at position −1 (i.e.the residue denoted as Xaa1 herein above), induce Erk1 (FIG. 2A) andErk2 (FIG. 2B) and total Erk phosphorylation (FIG. 2C). Open bars:controls; hatched bars: peptide induced Erk phosphorylation; black bars:peptide-induced Erk phosphorylation after pretreatment with the AT2receptor antagonist PD123319. At the bars (FIGS. 2A and 2B) the identityof the amino acid “X” at position −1 of XcAng-(1-7) is indicated.Pretreatment with PD123319 did not affect the ScAng-(1-7)-induced Erk2phosphorylation (FIG. 2B), nor the linear-DDRVAIHA (SEQ ID NO:4)-inducedtotal Erk phosphorylation (FIG. 2C). dKcAng-(1-7) is cAng-(1-7) with anN-terminal extension with a D-isomer of Lye.

FIGS. 3A and 3B. AT2 receptor stimulation is not exerted by cAng-(1-7)analogs with two amino acids N-terminal extension. FIG. 3A: DcAng-(1-7)and IcAng-(1-7) induced Erk1 phosphorylation of Erk1 in HBE cells. FIG.3B: IDcAng-(1-7) and IDDRVAIHA (SEQ ID NO:8)-induced total Erkphosphorylation in HBE cells. Open bar: control; hatched bars:peptide-induced ERK phosphorylation without pretreatment of the HBEcells; black bars: peptide-induced ERK phosphorylation after apretreatment with PD123319.

FIG. 4. N-extended cAng-(1-7) analogs do not act via the Mas receptor.The Mae receptor antagonist D-Pro7 does not inhibit the KcAng-(1-7)- andYcAng-(1-7)-induced vaseodilation of phenylephridine-precontracted ringsof aorta of Sprague Dawley rat. x: control; ◯: KcAng-(1-7); ∇:KcAng-(1-7) and Mas receptor antagonist D-Pro7; ●: YcAng-(1-7); ▾:YcAng-(1-7) and Mas receptor antagonist D-Pro7.

DETAILED DESCRIPTION

In view of the recent recognition of the AT2 receptor as an importantnew target for therapy, e.g. in the overall care of patients withhypertension, the present inventors set out to identify furthereffective and/or selective AT2 receptor agonists, which are expected tofind utility in inter alia the above-mentioned conditions. It wassurprisingly found that thioether-cyclized peptide analogs of Ang(1-7)extended with an additional amino acid at the N-terminus are potentagonists of the AT2 receptor. Accordingly, there is provided a cyclicpeptide compound consisting of the amino acid sequenceXaa¹-Asp-Arg-Ile/Val-Xaa⁵-Ile/Val-His-Xaa⁸ (SEQ ID NO:2) comprising athioether-bridge linkage between the side chains of Xaa⁵ and Xaa⁸ suchthat amino acids Xaa⁵ and Xaa⁸ together form a structure according toeither one of the general formula:

wherein R, R¹, R², R³, R⁴ and R⁵ are independently selected from —H, alower (e.g. C₁-C₁₀) alkyl or aralkyl group,

and wherein Xaa¹ is selected from the group consisting of charged aminoacids, aromatic amino acids and hydrophobic amino acids, andprotease-resistant variants thereof,

or a pharmaceutically acceptable salt thereof.

Peptide compounds and salts are referred to together hereinafter as “thecompounds of the invention”.

Pharmaceutically-acceptable salts include acid addition salts and baseaddition salts. Examples of such salts are acid addition salts formedwith inorganic acids, for example, hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid, and the like. Salts mayalso be formed with organic acids such as, for example, acetic acid,oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid,gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid,tannic acid, pamoic acid, alginic acid, polyglutamic acid, and the like.Salts may be formed with polyvalent metal cations such as zinc, calcium,bismuth, barium, magnesium, aluminum, copper, cobalt, nickel and thelike or with an organic cation formed from N,N′-dibenzylethylenediamineor ethylenediamine, or combinations thereof (e.g., a zinc tannate salt).The non-toxic, physiologically acceptable salts are preferred. Suchsalts may be formed by conventional means, for example by reaction of afree acid or a free base form of a compound of the invention with one ormore equivalents of an appropriate acid or base, optionally in asolvent, or in a medium in which the salt is insoluble, followed byremoval of said solvent, or said medium, using standard techniques (e.g.in vacuo or by freeze-drying). Salts may also be prepared by exchanginga counter-ion of a compound of the invention in the form of a salt withanother counter-ion, for example using a suitable ion exchange resin.

Preferably, R, R¹, R², R³, R⁴ and R⁵ are independently selected from Hand CH₃.

It was found that AT2 receptor interaction requires the presence of alanthionine ring linking the amino acids at positions 5 and 8 in theabove formula. Furthermore, the N-terminal extension should be limitedto a single amino acid residue as it was found that the addition of asecond additional residue abolished AT2 receptor interaction.

In one embodiment, the invention provides a peptide comprising athio-ether bridge according to formula A, i.e. wherein the linkagebetween the amino acids at positions 5 and 8 has the meaning—RCR¹—S—R²CR³—, wherein R, R¹, R² and R³ are independently selected from—H, a lower (e.g. C₁-C₁₀) alkyl or aralkyl group. R, R¹, R² and R³ arepreferably independently selected from H and CH₃. Peptides comprising athio-ether bridge according to formula A can be made for example bylantibiotic enzymes or by sulfur extrusion of a disulfide. The disulfidefrom which the sulfur is extruded can be formed by a D-cysteine inposition 5 and a L-cysteine in position 8 or by one D-cysteine inposition 5 and a L-penicillamine in position 8 [Galande, Trent andSpatola 2003 Biopolymers 71, 534-551].

Alternatively, the linkage of the two amino acids can be composed ofRCR¹—R²CR³—S—R⁴CR⁵ (Formula B) or RCR¹—S—R⁴CR⁵—R²CR³ (Formula C), inwhich R, R¹, R², R³, R⁴ and R⁵ independently represent —H, a lower (e.g.C₁-C₁₀) alkyl or aralkyl group. Peptides comprising a thio-ether bridgeaccording to formula B can for instance be made by sulfur extrusion of adisulfide formed by a D-homocysteine in position 5 and a L-cysteine inposition 8 [Galande, Trent and Spatola 2003 Biopolymers 71, 534-551].Likewise, peptides comprising a thio-ether bridge according to formula Ccan for instance be made by sulfur extrusion of a disulfide formed by aD-cysteine in position 5 and a L-homocysteine in position 8 [Galande,Trent and Spatola 2003 Biopolymers 71, 534-551].

It is convenient that a peptide analog of the invention can be made in abiological system, in particular making use of the lantibiotic enzymesystem of a (bacterial) host cell. Accordingly, peptides comprising athioether-bridge according to formula A are preferred.

In one aspect, Xaa¹ is a positively charged amino acid, preferably Lysor Arg or peptidase-resistant variant or derivate thereof. In anotheraspect, Xaa¹ is a negatively charged amino acid, preferably Asp or Gluor peptidase-resistant variant or derivate thereof. Alternatively Xaa¹is a hydrophobic amino acid, preferably Ile, Leu or Val, or an aromaticamino acid, preferably Tyr or Phe, or peptidase-resistant variant orderivative thereof. Peptidase-resistant variants and derivatives canprotect the analog against degradation by amino peptidase(s). In oneembodiment, Xaa¹ is a peptidase-resistant amino acid variant orderivate, for example one of the above mentioned amino acids in theD-configuration. Other suitable derivatives include those in which thefree amino group is cyclized to form a ring structure, likepyroglutamate (pGlu) wherein the terminus is a lactam ring.

The residues at positions 2, 3, 4, 6 and 7 can be varied as long asbiological activity is maintained. In a preferred embodiment, Xaa2 isAsp, Xaa3 is Arg, Xaa4 is Val, Xaa6 is Ile and/or Xaa 7 is His. Thestereochemistry of the residues involved in the thioether bridge can beeither the L- or D-form. Cyclic peptides wherein Xaa5 is aD-stereoisomer and/or Xaa8 is an L-stereoisomer are preferred. Mostpreferred is an analog wherein Xaa⁵ is a D-stereoisomer and Xaa⁸ is anL-stereoisomer.

Thioether-cyclized analogs of Angiotensin-(1-7) are known in the art.See for example Kluskens et al. (J Pharmacol Exp Ther. 2009 March;328(3):849-54) and WO2008/130217. Linear angiotensin analogs with orwithout N-terminal extensions of 1 to 3 amino acids have been describedby K. Rodgers et al., for example in WO99/40106, WO99/52540 andWO96/39164. However, a cAng(1-7) analog having an N-terminal extensionwith a single residue selected from the charged amino acids, aromaticamino acids and hydrophobic amino acids has not been taught or suggestedin the art.

According to specific embodiments of the invention, the cyclic peptidecompound is selected from the group consisting of:

Lys-Asp-Arg-Val-Abu/Ala-Ile-His-Abu/Ala (abbreviated to “K-cAng(1-7)”)(SEQ ID NO:3)

Asp-Asp-Arg-Val-Abu/Ala-Ile-His-Abu/Ala (abbreviated to“D-cAng(1-7)”)(SEQ ID NO:4)

Tyr-Asp-Arg-Val-Abu/Ala-Ile-His-Abu/Ala (abbreviated to“Y-cAng(1-7)”)(SEQ ID NO:5)

Ile-Asp-Arg-Val-Abu/Ala-Ile-His-Abu/Ala (abbreviated to“I-cAng(1-7)”)(SEQ ID NO:6)

Asn-Asp-Arg-Val-Abu/Ala-Ile-His-Abu/Ala (abbreviated to“N-cAng(1-7)”)(SEQ ID NO:7)

under the provision that the peptide does not contain two Abu(2-aminobutyric acid) residues. In view of their potency as potentvasodilating peptides, compounds K-cAng(1-7) and Y-cAng(1-7) areparticularly preferred.

Compounds of the invention are useful because they possesspharmacological activity. The compounds of the invention are thereforeindicated as pharmaceuticals. In particular, compounds of the inventionare agonists of the AT2 receptor, and, especially, are selectiveagonists of that sub-receptor, for example as demonstrated in the testsdescribed below. The compounds of the invention are expected to beuseful in those conditions where AT2 receptors are expressed and theirstimulation is desired or required.

The compounds of the invention are further indicated in the treatment ofconditions characterised by vasoconstriction, increased cell growthand/or differentiation, increased cardiac contractility, increasedcardiovascular hypertrophy, and/or increased fluid and electrolyteretention. Furthermore, the compounds of the invention are indicated inthe treatment of stress-related disorders, and/or in the improvement ofmicrocirculation and/or mucosa-protective mechanisms. Accordingly,compounds of the invention are expected to be useful in the treatment ofdisorders, which may be characterised as indicated above, and which areof, for example, the gastrointestinal tract, the cardiovascular system,the respiratory tract, the kidneys, the eyes, the female reproductive(ovulation) system and the central nervous system (CNS). Also providedis a method for accelerating tissue repair, comprising administering toa subject in need thereof a therapeutically effective dose of a peptidecompound of the invention.

Disorders of the gastrointestinal tract that may be mentioned includeoesophagitis, Barrett's oesophagus, gastric ulcers, duodenal ulcers,dyspepsia (including non-ulcer dyspepsia), gastro-oesophageal reflux,irritable bowel syndrome (IBS), inflammatory bowel disease (IBD),pancreatitis, hepatic disorders (such as hepatitis), gall bladderdisease, multiple organ failure (MOF) and sepsis. Other gastrointestinaldisorders that may be mentioned include xerostomia, gastritis,gastroparesis, hyperacidity, disorders of the bilary tract, coelicia,Crohn's disease, ulcerative colitis, diarrhoea, constipation, colic,dysphagia, vomiting, nausea, indigestion and Sjögren's syndrome.Disorders of the respiratory tract that may be mentioned includeinflammatory disorders, such as asthma, obstructive lung diseases (suchas chronic obstructive lung disease), pneumonitis, pulmonaryhypertension and acute respiratory distress syndrome. Disorders of thekidneys that may be mentioned include renal failure, nephritis and renalhypertension. Disorders of the eyes that may be mentioned includediabetic retinopathy, premature retinopathy and retinalmicrovascularisation. Disorders of the female reproductive system thatmay be mentioned include ovulatory dysfunction. Cardiovascular disordersthat may be mentioned include hypertension, cardiac hypertrophy, cardiacfailure, artherosclerosis, arterial thrombosis, venous thrombosis,endothelial dysfunction, endothelial lesions, post-balloon dilatationstenosis, angiogenesis, diabetic complications, microvasculardysfunction, angina, cardiac arrhythmias, claudication intermittens,preeclampsia, myocardial infarction, reinfarction, ischaemic lesions,erectile dysfunction and neointima proliferation. Disorders of the CNSthat may be mentioned include cognitive dysfunctions, dysfunctions offood intake (hunger/satiety) and thirst, stroke, cerebral bleeding,cerebral embolus and cerebral infarction. Peptide compounds of theinvention are also of use for augmenting erythropoiesis, e.g. bycontacting erythroid progenitor cells with at least one peptide compoundin an amount effective to augment erythropoiesis.

Compounds of the invention may also be useful in the modulation ofgrowth metabolism and proliferation, for example in the treatment ofhypertrophic disorders, prostate hyperplasia, autoimmune disorders,psoriasis, obesity, neuronal regeneration, the healing of ulcers,inhibition of adipose tissue hyperplasia, stem cell differentiation andproliferation, cancer (e.g. in the gastrointestinal tract, lung cancer,etc), apoptosis, tumours (generally) and hypertrophy, diabetes, neuronallesions and organ rejection.

The compounds of the invention are indicated both in the therapeuticand/or prophylactic treatment of the above conditions.

According to a further aspect of the invention there is thus providedthe compounds of the invention for use as pharmaceuticals. Also providedis a pharmaceutical composition comprising at least one peptide compoundof the invention and a pharmaceutically acceptable carrier and/oradjuvant. The pharmaceutical compositions contain the compoundsaccording to the invention in admixture with organic or inorganiccarriers suitable for enteral or parenteral administration. Thuspharmaceutical compositions may be formulated as solid lyophilizates, inwhich various inert compounds not reacting with peptides, e.g.,hydrocarbons can be used as carriers. When the pharmaceuticalcompositions are formulated as dilute or concentrated suspensions oremulsions, they contain also various preserving agents and stabilizingagents. Illustrative of the adjuvants which can be incorporated intablets, capsules and the like are the following: a binder such as gumtragacanth, acacia, corn starch or gelatin; an excipient such asmicrocrystalline cellulose; a disintegrating agent such as corn starch,pregelatinized starch, alginic acid and the like; a lubricant such asmagnesium stearate; a sweetening agent such as sucrose, lactose orsaccharin; a flavoring agent such as peppermint, oil of wintergreen orcherry. When the dosage unit form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier such as fattyoil. Various other materials may be present as coatings or to otherwisemodify the physical form of the dosage unit. For instance, tablets maybe coated with shellac, sugar or both. A syrup or elixir may contain theactive compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry ororange flavor.

According to a further aspect of the present invention, there isprovided a method of treatment of a condition in which endogenousproduction of AT2 receptor agonists is deficient, and/or a conditionwhere an increase in the effect of AT2 receptor agonists is desired orrequired, and/or a condition where AT2 receptors are expressed and theirstimulation is desired or required, which method comprisesadministration of a therapeutically effective amount of a compound ofthe invention to a person suffering from, or susceptible to, such acondition.

The compounds of the invention will normally be administered orally,intravenously, subcutaneously, buccally, rectally, dermally, nasally,tracheally, bronchially, by any other parenteral route or viainhalation, in a pharmaceutically acceptable dosage form.

When the condition to be treated is multiple organ failure, preferredroutes of administration are parenteral (e.g. by injection). Otherwise,the preferred route of administration for compounds of the invention isoral. The compounds of the invention may be administered alone, but arepreferably administered by way of known pharmaceutical formulations,including tablets, capsules or elixirs for oral administration,suppositories for rectal administration, sterile solutions orsuspensions for parenteral or intramuscular administration, and thelike. Such formulations may be prepared in accordance with standardand/or accepted pharmaceutical practice.

According to a further aspect of the invention there is thus provided apharmaceutical formulation including a compound of the invention, inadmixture with a pharmaceutically acceptable adjuvant, diluent orcarrier.

Compounds of the invention may also be administered in combination withother AT2 agonists that are known in the art, as well as in combinationwith AT1 receptor antagonists that are known in the art, such aslosartan, or in combination with an inhibitor of angiotensin convertingenzyme (ACE), or in combination with Mae receptor agonists e.g. 4,7cyclic angiotensin-(1-7) or angiotensin converting enzyme2 (ACE2).

In a specific aspect, the invention relates to use of compounds of theinvention in accelerating the growth or healing of tissue. For example,provided is a peptide compound according to the invention for use intissue repair. Also provided is a composition for accelerating woundhealing, comprising a suitable carrier or diluent and an amounteffective to accelerate wound healing of at least one peptide compoundof the invention. The compound can be administered in a matrical ormicellar solution. In one embodiment, the compound is administered at arate of at least 0.1 ng per kg body weight in a suitable carrier ordiluent. The carrier or diluent may be selected from the groupconsisting of carboxymethyl cellulose preparations, crystalloidpreparations, viscoelastics, polyethylene glycols and polypropyleneglycols. The compound can advantageously be administered in conjunctionwith a wound dressing.

According to a further aspect of the invention, there is provided acombination product comprising (A) a peptide compound of the invention;and (B) selected from an AT1 receptor antagonist or an ACE inhibitor,and/or (C) a Mas receptor agonist, or ACE2, wherein each of components(A), (B) and/or (C) is formulated in admixture with apharmaceutically-acceptable adjuvant, diluent or carrier. Suchcombination products provide for the administration of compound of theinvention in conjunction with an AT1 receptor antagonist, or an ACEinhibitor, a Mas receptor agonist, or ACE2 and may thus be presentedeither as separate formulations, wherein at least one of thoseformulations comprises compound of the invention, and at least onecomprises for instance AT1 receptor antagonist, or ACE inhibitor, or maybe presented (i.e. formulated) as a combined preparation (i.e. presentedas a single formulation including compound of the invention and AT1receptor antagonist or ACE inhibitor).

Thus, there is further provided: (1) a pharmaceutical formulationincluding a compound of the invention and an AT1 receptor antagonist, oran ACE inhibitor or a Mas receptor agonist, or ACE2, in admixture with apharmaceutically-acceptable adjuvant, diluent or carrier; and

(2) a kit of parts comprising components:

(a) a pharmaceutical formulation including a compound of the invention,in admixture with a pharmaceutically-acceptable adjuvant, diluent orcarrier; and

(b) a pharmaceutical formulation including an AT1 receptor antagonist,or an ACE inhibitor,

(c) a Mas receptor agonist, or ACE2 in admixture with a pharmaceuticallyacceptable adjuvant, diluent or carrier, which components (a), (b) and(c) are each provided in a form that is suitable for administration inconjunction with the other.

Depending upon the disorder and patient to be treated and the route ofadministration, the compounds of the invention may be administered atvarying doses.

Although doses will vary from patient to patient, suitable doses e.g.for subcutaneous administration are in the range of about 1-1000microgram/kg/day per patient, administered in single doses. Morepreferred daily doses are in the range 40 to 80 microgram/kg/day perpatient. Intravenous doses are identical to subcutaneous doses.Pulmonary doses are about 4-fold higher than subcutaneous doses. Oraldoses are at least five fold higher than subcutaneous doses and stronglydepend on the applied formulation. Compounds of the invention can alsobe administered once per week. Weekly doses are about 10- to 30-foldhigher than daily doses.

In any event, the physician, or the skilled person, will be able todetermine the actual dosage which will be most suitable for anindividual patient, which is likely to vary with the condition that isto be treated, as well as the age, weight, sex and response of theparticular patient to be treated. The above-mentioned dosages areexemplary of the average case; there can, of course, be individualinstances where higher or lower dosage ranges are merited, and such arewithin the scope of this invention. Compounds of the invention have theadvantage that they bind selectively to, and exhibit agonist activityat, the AT2 receptor. By compounds which “bind selectively” to the AT2receptor, we include that the affinity ratio for the relevant compound(AT2:AT1) is at least 5:1, preferably at least 10:1 and more preferablyat least 20:1. The compounds of the invention may also have theadvantage that they may be more efficacious than, be less toxic than, belonger acting than, be more potent than, produce fewer side effectsthan, be more easily absorbed than, and/or have a better pharmacokineticprofile (e.g. higher oral bioavailability and/or lower clearance) than,and/or have other useful pharmacological, physical, or chemicalproperties over, compounds known in the prior art.

EXAMPLES Example 1. KcAng-(1-7) and YcAng-(1-7) are Potent Vasodilators

This example demonstrates that N-extended cAng-(1-7) analogs canstrongly induce vasodilation in precontracted aorta rings from SpragueDawley rats.

Materials and Methods.

Peptides: KDRVdCIHC, and YDRVdCIHC wherein dC stands for D-cysteine werepurchased from JPT. Specified pathogen-free male Sprague Dawley rats(SD) (Harlan, Zeist, The Netherlands), weighing 350-450 gram were used.Prior to the experiment, the animals were housed together with freeaccess of tap water and solid chow (Harlan, Zeist, The Netherlands) in atemperature and humidity controlled room and a 12/12 h light/dark cycle.All protocols described were approved by the University of GroningenCommittee for Animal Experimentation.

Thioether-bridged peptides were obtained from the disulfide bridgedpeptide via base-assisted sulfur extrusion as previously (Galande A K,et al 2003. Biopolymers 71:534-6551).

Vasodilation on precontracted aorta-rings. Arterial rings for organ bathexperiments were prepared as described previously (Kluskens L D, et al.2009. J Pharmacol Exp Ther 328:849-54). Before testing the vasodilatingeffects of the peptides the rings were precontracted to 50% of theirmaximum contraction level with 30 nM phenylephrine (PE). The 5,8thioether bridged peptides KDRV[AIHA]_(c) (SEQ ID NO:3) orYDRV[AIHA]_(c) (SEQ ID NO:5) were added cumulatively in a range of 0.1nM to 1 μM. Vasodilation data are represented as percentage ofrelaxation of 30 nM PE contraction.

Results.

FIG. 1 demonstrates the capacity of KcAng-(1-7) and YcAng-(1-7) toinduce vasodilation of precontracted aorta rings from Sprague Dawlyrats. Both peptides had already at sub-nM concentration significantvasodilating capacity.

Conclusion.

These data demonstrate that KcAng-(1-7) and YcAng-(1-7) are highlypotent vasodilating peptides.

Example 2. N-Extended cAng-(1-7) Analogs Require the 4,7 Thioether Ringfor Agonistic Action Via the AT2 Receptor

Introduction.

This example shows the capacity of thioether-bridged cAng-(1-7), ifextended with one N-terminal amino acid, to stimulateextracellular-signal-regulated kinase (ERK) phosphorylation in humanbronchial epithelial (HBE) cells. The AT2 receptor specificity of theabove thioether bridged peptides is established by measuring whether ornot the peptide-induced ERK phosphorylation could be inhibited byPD123319, a well known antagonist of the AT2 receptor. In addition it isdemonstrated that the thioether ring in the N-terminally extendedanalogs is required for the agonistic action via the AT2 receptor.

Materials and Methods.

Peptides were purchased from JPT: linear DDRVAIHA (SEQ ID NO:4),dKDRVdCIHC, KDRVdCIHC, DDRVdCIHC, IDRVdCIHC, NDRVdCIHC, SDRVdCIHC, inwhich dK stands for D-lysine and dC stands for D-cysteine. The AT2receptor antagonist PD123319 was from Axon Medchem. Studies wereperformed using the immortal human bronchial epithelial cell line HBE(Cozens A L, et al. 1994 CFTR expression and chloride secretion inpolarized immortal human bronchial epithelial cells. Am J Respir CellMol Biol 10:38-47). All culture media and supplements, multiwell platesand flasks were from PAA.

Cell culture. Multi-well plates received 1 ml from a coating solution(for 112 ml: F12 medium 100 ml; bovine serum albumin 10 ml (1 mg/ml);collagen I, Bovine 1 ml (3 mg/ml); human fibronectin 1 ml (1 mg/ml)) andwere incubated for 6 hours at 37° C. and 5% CO₂. Subsequently thecoating solution was discarded and the plates were dried for 2 hours inthe laminar flow cabinet. In each 12 well plate 8·10⁴ HBE cells wereseeded per well followed by incubation for 24 hours at 37° C. and 5%CO₂. Cell culture medium was prepared by adding 432.2 ml MEM 199 basicmedium to 50 ml (15%) FCS and 5 ml L-Glutamin (200 mM), 10 mlPenicillin/Streptomycin (10.000 unite/ml/10 mg/ml) and 2.8 ml Gentamycin(10 mg/ml). At 85-90% confluency medium was replaced by medium withoutFCS and growth factors followed by again 24 hours incubation at 37° C.and 5% CO₂.

Erk1 and Erk2 phosphorylation. The medium was removed from each well andwas washed once with HBS buffer without calcium and magnesium. Thecapacity of peptide at 10⁻⁶ M was tested after 20 min preincubation witheither 10⁻⁶ M of PD123319 or concomitant controls, followed by washing.Challenges and concomitant controls were performed at 1 ml/well during10 min. All samples were tested in triplicate. After the challenge 150μl RIPA buffer (RIPA, TBS, 1% Nonidet P-40, 0.5% sodium deoxycholate,0.1% SDS, 0.004% sodium azide, 1% PMSF (2 mM final concentration), 1%sodium orthovanadate (1 mM final concentration) and 1% proteaseinhibitor cocktail) was added to each well. The plates were incubatedfor 15 minutes at 4° C. The lysed cells were scraped from the surface.The lysate was transferred to 1.5 ml eppendorf cups and centrifuged 5minutes at 12.700×g to pellet the cell debris. The supernatant wastransferred into new cups and of a 5 μl sample the total protein contentwas determined via the DC protein assay according to lowry (Bio-Rad),with BSA used as calibration protein. Protein was separated on gel andblotted with p-ERK mouse monoclonal IgG2a and mouse GAPDH anti-rat IgG1antibodies and as a second antibody goat anti-mouse IgG-AP. Thephosphorylated Erk proteins and GAPDH were stained with BCIP/NBT andquantified relative to GAPDH and total Erk using TotalLab software.Alternatively, total Erk phosphorylation was measured afterpermeabilization of the non-detached cells.

Results.

FIGS. 2A and 2B shows that cAng-(1-7) analogs which are extended withone amino acid at the N-terminus stimulate ERK1 and to a lesser extentERK2 phosphorylation. ERK1 and ERK2 phosphorylation could be inhibitedby the AT2 antagonist PD123319. The extent by which the AT2 antagonistPD123319 could inhibit the peptide-induced ERK phosphorylation wasdependent on the amino acid at the N-terminally extended position “−1”.Inhibition of ERK phosphorylation by PD123319 was complete in the casesof Asp, Lys and Ile at the extended position. When Asn or Ser werepresent at the extended position the inhibition of ERK1 phosphorylationwas less and inhibition of ERK2 phosphorylation was also less or absent.FIG. 2C demonstrates that the linear peptide DDRVAIHA (SEQ ID NO:4)induced Erk phosphorylation, but for this linear peptide the induced Erkphosphorylation could not be inhibited by PD123319. On the other hand,Erk phosphorylation induced by the thioether bridged dKcAng-(1-7) couldbe inhibited by PD123319.

Conclusions.

These data clearly prove that cAng-(1-7) with a single amino acidN-terminal extension stimulate ERK phosphorylation via the AT2 receptor.The interaction with the AT2 receptor of the N-terminally extendedcAng-(1-7) depends on the amino acid at position at the very N-terminus“−1”. At this N-terminal position “−1”, denoted as Xaa¹ elsewhere, apositively or negatively charged amino acid or a hydrophobic amino acidled to high AT2 receptor specificity; a peptide extended with ahydrophilic amino acid without net charge such as Ser and Asn did not orhardly interact with the AT2 receptor. The thioether ring is requiredfor the action of the N-extended cAng-(1-7) peptides via the AT2receptor, since the AT2 antagonist PD123319 could not inhibit the Erkphosphorylation induced by a linear N-extended Ang-(1-7) analog.

Example 3. AT2 Receptor Stimulation is not Exerted by cAng-(1-7) Analogswith Two Amino Acids N-Terminal Extension

Introduction.

In this example it is investigated whether or not cAng-(1-7) analogswith an N-terminal extension of two amino acids stimulate the AT2receptor. As in Example 2, peptide-induced ERK phosphorylation in HBEcells was measured and the dependence of this phosphorylation on the AT2antagonist PD123319.

Materials and Methods.

Thioether bridged peptide synthesis and cell culture and measurement ofErk1- and total Erk phosphorylation were essentially as in example 2.

Results.

FIG. 3A shows that both the thioether-bridged analog IcAng-(1-7) andDcAng-(1-7) induced Erk1 phosphorylation, which is inhibited by thepretreatment with PD123319. However, in the case of a two-amino acidextension as in IDcAng-(1-7)- and in the case of the linear IDDRVAIHA(SEQ ID NO:6)-induced Erk phosphorylation, no inhibition at all bypretreatment with the AT2 antagonist PD123319 was obtained (FIG. 3B).

Conclusions.

Example 2 had already demonstrated that a ring was required for the AT2receptor stimulation of N-extended Ang-(1-7). Consistently also theextension with two amino acids of a linear variant did not stimulate theAT2 receptor. Since the nonlinear cAng-(1-7) N-terminally extended withtwo amino acids did neither stimulate the AT2 receptor, it is concludedthat one amino acid N-terminal extension of cAng-(1-7) is the N-terminalextension limit for AT2 receptor agonists.

Example 4. N-Extended cAng-(1-7) Analogs do not Act Via the Mas Receptor

Introduction.

In this example it is demonstrated that the Mas receptor antagonistD-Pro7-Ang-(1-7) can not inhibit the KcAng-(1-7) and YcAng-(1-7)-inducedvasodilation in precontracted aorta rings from Sprague Dawley rats.

Materials and Methods.

D-Pro7-ang-(1-7), ang-(1-7) with a D-isomer of proline at position 7,which is a Mas-receptor antagonists, was from JPT and purified by HPLCprior to use. This antagonist was used as in Kluskens et al 2009: 10 minbefore addition of 30 nM PE at a concentration of 0.1 μM. Othermaterials and methods were as in example 1.

Results.

FIG. 4 demonstrates that the Mas receptor antagonist D-Pro7 does notinhibit the KcAng-(1-7) and YcAng-(1-7)-induced vasodilation inprecontracted aorta rings from Sprague Dawley rats.

Conclusion.

KcAng-(1-7) and YcAng-(1-7) do not act via de Mas receptor.

The invention claimed is:
 1. A cyclic peptide compound consisting of theamino acid sequence Xaa¹-Asp-Arg-Ile/Val-Xaa⁵-Ile/Val-His-Xaa⁸comprising a thioether-bridge linkage between the side chains of Xaa⁵and Xaa⁸ such that Xaa⁵ and Xaa⁸ together form a structure according toone of the general formula:

wherein R, R¹, R², R³, R⁴ and R⁵ are independently selected from thegroup consisting of —H and a C₁-C₁₀ alkyl or aralkyl group, and whereinXaa¹ is selected from the group consisting of charged amino acids,aromatic amino acids and hydrophobic amino acids and protease-resistantvariants thereof selected from D-stereoisomer or cyclized residue, or apharmaceutically acceptable salt thereof.
 2. The peptide compoundaccording to claim 1, wherein Xaa¹ is selected from the group consistingof protease-resistant variants of charged amino acids, aromatic aminoacids and hydrophobic amino acids.
 3. The peptide compound according toclaim 2, wherein Xaa¹ is a D-stereoisomer.
 4. The peptide compoundaccording claim 1, wherein Xaa¹ is a positively charged amino acid. 5.The peptide compound according to claim 4, wherein Xaa¹ is Lys or Arg.6. The peptide compound according to claim 1, wherein Xaa¹ is anegatively charged amino acid.
 7. The peptide compound according toclaim 6, wherein Xaa¹ is Asp, Glu or pyroGlu.
 8. The peptide compoundaccording to claim 1, wherein Xaa¹ is a hydrophobic amino acid.
 9. Thepeptide compound according to claim 8, wherein Xaa¹ is Ile, Leu or Val.10. The peptide compound according to claim 1, wherein Xaa¹ is anaromatic amino acid.
 11. The peptide compound according to claim 10,wherein Xaa¹ is Tyr or Phe.
 12. The peptide compound according to claim1, wherein Xaa⁵ is a D-stereoisomer.
 13. The peptide compound accordingto claim 1, wherein Xaa⁸ is an L-stereoisomer.
 14. The peptide compoundaccording to claim 1, wherein Xaa⁵ is a D-stereoisomer and Xaa⁸ is anL-stereoisomer.
 15. The peptide compound according to claim 1, whereinthe peptide compound is selected from the group consisting of:Lys-Asp-Arg-Val-Abu/Ala-Ile-His-Abu/Ala(K-cAng(1-7))(SEQ ID NO:3),Asp-Asp-Arg-Val-Abu/Ala-Ile-His-Abu/Ala(D-cAng(1-7))(SEQ ID NO:4),Tyr-Asp-Arg-Val-Abu/Ala-Ile-His-Abu/Ala (Y-cAng(1-7)) (SEQ ID NO:5),Ile-Asp-Arg-Val-Abu/Ala-Ile-His-Abu/Ala (I-cAng(1-7))(SEQ ID NO:6) andAsn-Asp-Arg-Val-Abu/Ala-Ile-His-Abu/Ala (N-cAng(1-7))(SEQ ID NO:7),under the provision that the peptide does not contain two Abu(2-aminobutyric acid) residues.
 16. Pharmaceutical compositioncomprising a peptide compound according to claim 1, and apharmaceutically acceptable adjuvant, diluent or carrier.
 17. An invitro method of activating angiotensin II type 2 (AT2) receptor, whereinthe method comprises contacting the AT2 receptor with a peptide compoundaccording to claim
 1. 18. The peptide compound according to claim 1 forthe treatment of a condition in which selective agonism of the AT2receptor is desired and/or required, wherein the condition is diabetes,myocardial infarction, renal hypertension, stroke, alopecia, aninflammatory lung disorder, fibrosis, or wherein said condition requiresaugmentation of erythropoiesis.
 19. A method for treatment of a subjecthaving a condition in which selective agonism of the AT2 receptor isdesired and/or required, comprising administering to the subject aneffective amount of the peptide compound of claim 1, wherein saidcondition is diabetes, myocardial infarction, renal hypertension,stroke, alopecia, an inflammatory lung disorder, fibrosis, or whereinsaid condition requires augmentation of erythropoiesis.
 20. Acombination product comprising (A) a peptide compound according to claim1; and (B) a compound selected from AT1 receptor antagonists or ACEinhibitors, and/or (C) a compound selected from Mas receptor agonists orACE2, wherein each of components (A), (B) and/or (C) is formulated inadmixture with a pharmaceutically-acceptable adjuvant, diluent orcarrier.